Devices and methods for detecting human metapneumovirus

ABSTRACT

The present invention discloses specific human metapneumovirus monoclonal antibodies. The antibody is at least two-fold less reactive with non-human metapneumoviruses including, but not limited to, respiratory viruses or avian metapneumoviruses. Further, the antibody is at least two-fold more reactive with a human metapneumovirus (i.e., for example, Type A or Type B) than with non-human metapneumoviruses including, but not limited to, respiratory viruses or avian metapneumoviruses. Consequently, these novel antibodies are useful as a clinical diagnostic agent, especially when using fresh nasopharengeal aspirates. The invention also contemplates numerous diagnostic platforms that together with the novel antibodies can support economical, fast, and highly selective detection and identification of clinical inoculum samples.

FIELD OF INVENTION

This invention is related to the detection and identification ofviruses. In one embodiment, the virus comprises a metapneumovirus. Inone embodiment, the virus infects a mammal. In one embodiment, theinfection may be identified using an antibody. In particular, theantibodies are monoclonal antibodies produced from a hybridoma cellculture.

BACKGROUND

The human metapneumovirus (hMPV) had been present in the humanpopulation for at least 50 years before it was first identified. NatureMedicine 7:P719-724 (2001). hMPV belongs to the Paramyxoviridae familyof viruses, which includes several well known human pathogens such asmeasles virus, mumps virus, parainfluenza viruses and the humanrespiratory syncytial virus (hRSV). Based on hMPV's genetic sequence andstructure, hMPV falls in the Pneumovirinae sub-family, together with itsclosest known human-infecting relative, Respiratory Syncytial Virus(RSV). However, hMPV's genetic sequence and structure differssufficiently from that of RSV and is consequently placed in a separategenus—the Metapneumoviruses.

hMPV infects people of all ages and causes mild to severe respiratoryinfections. By the age of 5, most children have been infected with hMPVat least once. Severe disease requiring hospitalization occurs primarilyin young children, the elderly and the immunocompromised. hMPV'sclinical impact and epidemiology is very similar to that of RSV andinfection by these two viruses cannot be distinguished on the bases ofclinical signs alone.

Human metapneumovirus is common worldwide and seems to be most active inlate winter and early spring—a period when many other respiratoryviruses are also circulating. Several epidemiological surveys on hMPVinfection have documented cases of metapneumovirus in Europe, Africa,Asia/Australia, Southern America, and Northern America. Worldwide, hMPVaccounts for a significant portion of respiratory tract illnesses inhospitalized children, with high incidences occurring during the wintermonths in moderate climate zones and late spring-early summer in thesubtropics. hMPV accounts for roughly 5 to 15% of the respiratory tractillnesses in hospitalized young children, with children <2 years of agebeing most at risk for serious hMPV infections. hMPV infections, likeRSV and influenza virus infections, also account for respiratory tractinfections in the elderly population and in patients with underlyingdisease.

What is needed is a simple, fast, and economical method to detect andidentify types and subtypes of human metapneumovirus without theimplementation of cell culture protocols, and without cross-reactivityto avian metapneumoviruses.

SUMMARY

This invention is related to the detection and identification ofviruses. In one embodiment, the virus comprises a metapneumovirus. Inone embodiment, the virus infects a mammal. In one embodiment, theinfection may be identified using an antibody. In particular, theantibodies are monoclonal antibodies produced from a hybridoma cellculture.

In one embodiment, the present invention contemplates a composition,comprising: a) a monoclonal antibody having specificity for a humanmetapneumovirus, wherein said antibody is at least two-fold lessreactive with a virus selected from the group consisting of respiratoryvirus and avian metapneumovirus; and b) a binding partner, wherein saidpartner is capable of interacting with said antibody. In one embodiment,the antibody is specific for Type A human metapneumovirus. In oneembodiment, the antibody is specific for Type B human metapneumovirus.In one embodiment, the binding partner comprises an epitope selectedfrom the group consisting of protein L, protein N, protein F, andprotein P. In one embodiment, the binding partner comprises an epitopeselected from the group consisting of protein L, protein N, protein F,and protein P. In one embodiment, the binding partner is a protein. Inone embodiment, the binding partner is a nucleic acid. In oneembodiment, the protein is a protein derived from a humanmetapneumovirus.

In one embodiment, the present invention contemplates a composition,comprising: a) a monoclonal antibody having specificity for a humanmetapneumovirus, wherein said antibody is at least two-fold morereactive with said human metapneumovirus than a second virus selectedfrom the group consisting of respiratory virus and avianmetapneumovirus; and b) a binding partner, wherein said partner iscapable of interacting with said antibody under conditions that is atleast two-fold more reactive than a second virus selected from the groupcomprising respiratory virus or avian metapneumovirus. In oneembodiment, the antibody is specific for Type A human metapneumovirus.In one embodiment, the antibody is specific for Type B humanmetapneumovirus. In one embodiment, the binding partner comprises anepitope selected from the group consisting of protein L, protein N,protein F, and protein P. In one embodiment, the binding partnercomprises an epitope selected from the group consisting of protein L,protein N, protein F, and protein P. In one embodiment, the bindingpartner is a protein. In one embodiment, the binding partner is anucleic acid. In one embodiment, the protein is a protein derived from ahuman metapneumovirus.

In one embodiment, the present invention contemplates a panel,comprising a first and second monoclonal antibodies to humanmetapneumovirus, wherein said antibodies are at least two-fold lessreactive with a second virus selected from the group consisting ofrespiratory virus and avian metapneumovirus. In one embodiment, thefirst antibody is specific for Type A human metapneumovirus. In oneembodiment, the second antibody is specific for Type B humanmetapneumovirus. In one embodiment, the first antibody binds to anepitope selected from the group consisting of protein L, protein N,protein F, and protein. P. In one embodiment, the second antibody bindsto an epitope selected from the group consisting of protein L, proteinN, protein F, and protein P.

In one embodiment, the present invention contemplates a monoclonalantibody having specificity for a human metapneumovirus, wherein saidantibody is at least two-fold less reactive with a second virus selectedfrom the group consisting of respiratory virus and avianmetapneumovirus. In one embodiment, the antibody is specific for Type Ahuman metapneumovirus. In one embodiment, the antibody is specific forType B human metapneumovirus. In one embodiment, the antibody binds toan epitope selected from the group consisting of protein L, protein N,protein F, and protein P. In one embodiment, the antibody binds to anepitope selected from the group consisting of protein L, protein N,protein F, and protein P.

In one embodiment, the present invention contemplates a monoclonalantibody having specificity for a human metapneumovirus, wherein saidantibody is at least two-fold more reactive with said humanmetapneumovirus than a second virus selected from the group consistingof respiratory virus and avian metapneumovirus. In one embodiment, theantibody is specific for Type A human metapneumovirus. In oneembodiment, the antibody is specific for Type B human metapneumovirus.In one embodiment, the antibody binds to an epitope selected from thegroup consisting of protein L, protein N, protein F, and protein P. Inone embodiment, the antibody binds to an epitope selected from the groupconsisting of protein L, protein N, protein F, and protein P.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) an animal comprising spleen cells; ii) aninoculum comprising human metapneumovirus; and iii) a cultured cellcapable of fusing with said spleen cells; b) immunizing said animal withsaid inoculum so as to create an immunized animal; c) isolating at leasta portion of said spleen cells from said immunized animal; d) fusingsaid spleen cell with said cultured cell such that a hybridoma cellculture is produced, wherein said cell culture produces a monoclonalantibody having reactivity with said human metapneumovirus, wherein saidantibody is at least two-fold less reactive with a second virus selectedfrom the group consisting of respiratory virus and avianmetapneumovirus, or preferably, said antibody is two-fold more reactivewith said human metapneumovirus than with said second virus selectedfrom the group comprising respiratory virus or avian metapneumovirus. Inone embodiment, the animal is a mouse. In one embodiment, the humanmetapneumovirus comprises Type A human metapneumovirus. In oneembodiment, the human metapneumovirus comprises Type B humanmetapneumovirus. In one embodiment, the inoculum comprises anasopharangeal aspirate. In one embodiment, the aspirate is collectedfrom a human. In one embodiment, the antibody is specific for Type Ahuman metapneumovirus. In one embodiment, the antibody is specific forType B human metapneumovirus. In one embodiment, the antibody binds toan epitope selected from the group consisting of protein L, protein N,protein F, and protein P.

DEFINITIONS

The term “antibody”, as used herein, refers to any polypeptidesubstantially encoded by an immunoglobulin gene or immunoglobulin genes,or fragments thereof which specifically bind and recognize an analyte(antigen). The recognized immunoglobulin genes include the kappa,lambda, alpha, gamma, delta, epsilon and mu constant region genes, aswell as the myriad immunoglobulin variable region genes. Light chainsare classified as either kappa or lambda. Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Anantibody may be of animal (especially mouse or rat) or human origin ormay be chimeric or humanized.

The term “hMPV antibody”, as used herein, refers to any antibody orantibody fragment that specifically binds a polypeptide encoded by thehuman metapneumovirus genome, cDNA, or a subsequence thereof, or aglycolipid (i.e., a lipid comprising an attached sugar residue).

The term “susceptible to infection”, as used herein, refers to theability of a cell to become infected with virus or another intracellularorganism. Although it encompasses “permissive” infections, it is notintended that the term be so limited, as it is intended that the termencompass circumstances in which a cell is infected, but the organismdoes not necessarily replicate and/or spread from the infected cell toother cells. The phrase “viral proliferation,” as used herein describesthe spread or passage of infectious virus from a permissive cell type toadditional cells of either a permissive or susceptible character.

The term, “humanized antibody”, as used herein, refer to any chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies).Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fe), typically that of a humanimmunoglobulin. Jones et al., Nature, 321:522-525 (1986). Generally, ahumanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. Accordingly, such “humanized”antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567; hereinincorporated by reference), wherein substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a non-human species.

The term “chimeric antibody”, as used herein, refers to any antibodymolecule in which (a) the constant region, or a portion thereof, isaltered, replaced or exchanged so that the antigen binding site(variable region) is linked to a constant region of a different oraltered class, effector function and/or species, or an entirelydifferent molecule which confers new properties to the chimericantibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or(b) the variable region, or a portion thereof, is altered, replaced orexchanged with a variable region having a different or altered antigenspecificity.

The term “epitope”, as used herein, refers to any molecular region onthe surface of an antigen capable of eliciting an immune response and ofcombining with the specific antibody produced by such a response. Forexample, an antigen may comprise amino acids (i.e., for example, aprotein or peptide) or nucleic acids (i.e., for example, an oligonucleicacid, ribonucleic acid etc.)

The term “immunoassay”, as used herein, refers to any assay that uses anantibody to specifically bind an analyte. The immunoassay ischaracterized by the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the analyte.

The phrase “specifically (or selectively) binds to an antibody” or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, as used herein, refers to any binding reaction thatis determinative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein under conditions such that any binding to other proteins presentin the sample is at least two-fold less, preferably at least ten-foldless, and more preferably one hundred-fold less. Specific binding to anantibody under such conditions may require an antibody that is selectedfor its specificity for a particular protein. For example, antibodiesraised an hMPV can be selected to obtain antibodies specificallyimmunoreactive with that protein and not with other proteins, except forpolymorphic variants. A variety of immunoassay formats may be used toselect antibodies specifically immunoreactive with a particular protein.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

The term “inoculating suspension,”, “inoculum, or “inoculant”, as usedherein, refers to any suspension which may be inoculated with organismsto be tested. It is not intended that the term “inoculating suspension,”be limited to a particular fluid or liquid substance. For example,inoculating suspensions may be comprised of water, saline, or an aqueoussolution. It is also contemplated that an inoculating suspension mayinclude a component to which water, saline or any aqueous material isadded. It is contemplated in one embodiment, that the componentcomprises at least one component useful for the intended microorganism.It is not intended that the present invention be limited to a particularcomponent.

The terms “sample” and “specimen”, as used herein, include anycomposition that is obtained and/or derived from biological orenvironmental source, as well as sampling devices (e.g., swabs) whichare brought into contact with biological or environmental samples.“Biological samples” include those obtained from an animal (includinghumans, domestic animals, as well as feral or wild animals, such asungulates, bear, fish, lagamorphs, rodents, etc.), body fluids such asurine, blood, fecal matter, cerebrospinal fluid (CSF), semen, sputum,and saliva, as well as solid tissue. Also included are samples obtainedfrom food products and food ingredients such as dairy items, vegetables,meat, meat by-products, and waste. “Environmental samples” includeenvironmental material such as surface matter, soil, water, andindustrial materials, as well as material obtained from food and dairyprocessing instruments, apparatus, equipment, disposable, andnon-disposable items. These examples are not to be construed as limitingthe sample types applicable to the present invention.

The term “culture”, as used herein, refers to any composition, whetherliquid, gel, or solid, which contains one or more microorganisms and/orone or more cells. A culture of organisms and/or cells can be pure ormixed. For example, a “pure culture” of an organism as used hereinrefers to a culture in which the organisms present are of only onestrain of a single species of a particular genus. This is in contrast toa “mixed culture” of organisms which refers to a culture in which morethan one strain of a single genus and/or species of microorganism ispresent.

The terms “culture media,” and “cell culture media,” as used herein,refer to any media that are suitable to support maintenance and/orgrowth of cells in vitro (i.e., cell cultures).

The term “animal”, as used herein, refers to any organism that iscapable of becoming immunized by a viral particle (i.e., for example, anhMPV viral particle). For example, an animal may include, but is notlimited to, human, dog, cat, cattle, sheep, mice, rats, goats etc.

The term “primary cell”, as used herein, refers to any cell which isdirectly obtained from a tissue or organ of a host whether or not thecell is in culture.

The term “cultured cell”, as used herein, refers to any cell which hasbeen maintained and/or propagated in vitro. Cultured cells includeprimary cultured cells and cell lines.

The term “primary cultured cells”, as used herein, refers to any primarycells which are in in vitro culture and which preferably, though notnecessarily, are capable of undergoing ten or fewer passages in in vitroculture before senescence and/or cessation of proliferation.

The terms “cell line” and “immortalized cell”, as used herein, refers toany cell which is capable of a greater number of cell divisions in vitrobefore cessation of proliferation and/or senescence as compared to aprimary cell from the same source. A cell line includes, but does notrequire, that the cells be capable of an infinite number of celldivisions in culture. The number of cell divisions may be determined bythe number of times a cell population may be passaged (i.e.,subcultured) in in vitro culture. Passaging of cells is accomplished bymethods known in the art. Briefly, a confluent or subconfluentpopulation of cells which is adhered to a solid substrate (e.g., plasticPetri dish) is released from the substrate (e.g., by enzymaticdigestion), and a proportion (e.g., 10%) of the released cells is seededonto a fresh substrate. The cells are allowed to adhere to thesubstrate, and to proliferate in the presence of appropriate culturemedium. The ability of adhered cells to proliferate may be determinedvisually by observing increased coverage of the solid substrate over aperiod of time by the adhered cells. Alternatively, proliferation ofadhered cells may be determined by maintaining the initially adheredcells on the solid support over a period of time, removing and countingthe adhered cells and observing an increase in the number of maintainedadhered cells as compared to the number of initially adhered cells.

The terms “cytopathic effect” or “CPE” as used herein, describe changesin cellular structure (i.e., a pathologic effect). Common cytopathiceffects include cell destruction, syncytia (i.e., fused giant cells)formation, cell rounding, vacuole formation, and formation of inclusionbodies. CPE results from actions of a virus on permissive cells thatnegatively affect the ability of the permissive cellular host to preformits required functions to remain viable. In in vitro cell culturesystems, CPE is evident when cells, as part of a confluent monolayer,show regions of non-confluence after contact with a specimen thatcontains a virus. The observed microscopic effect is generally focal innature and the foci are initiated by a single virion. However, dependingupon viral load in the sample, CPE may be observed throughout themonolayer after a sufficient period of incubation. Cells demonstratingviral induced CPE usually change morphology to a rounded shape, and overa prolonged period of time can die and be released form their anchoragepoints in the monolayer. When many cells reach the point of focaldestruction, the area is called a viral plaque, which appears as a holein the monolayer. The terms “plaque” and “focus of viral infection”refer to a defined area of CPE which is usually the result of infectionof the cell monolayer with a single infectious virus which thenreplicates and spreads to adjacent cells of the monolayer. Cytopathiceffects are readily discernable and distinguishable by those skilled inthe art.

The term “hybridoma(s),” as used herein, refers to cells produced byfusing two cell types together. Commonly used hybridomas include thosecreated by the fusion of antibody-secreting B cells from an immunizedanimal, with a malignant myeloma cell line capable of indefinite growthin vitro. For example, a spleen cell from an immunized animal may befused together with Ps2/0Ag14 myeloma cell that can be cloned and usedto prepare monoclonal antibodies (i.e., for example, hMPV MAbs).

A “target analyte” is any molecule or molecules that are to be detectedand/or quantified in a sample. For example, target analytes may includebiomolecules including, but not limited to, nucleic acids, antibodies,proteins, sugars, and the like. In particular, a target analyte wouldinclude a human metapneumovirus protein and/or nucleic acid.

The terms “binding partner” or “member of a binding pair” refer tomolecules that specifically bind other molecules to form a bindingcomplex such as antibody-antigen, lectin-carbohydrate, nucleicacid-nucleic acid, biotin-avidin, etc. The binding complex ispredominantly mediated by non-covalent (e.g. ionic, hydrophobic, etc.)interactions. The terms “binding partner” and “member of a binding pair”apply to individual molecules, as well as to a set of multiple copies ofsuch molecules, e.g., affixed to a distinct location of a surface. Thus,as used herein, the expression “different binding partners” includessets of different binding partners, wherein each set includes multiplecopies of one type of binding partner which differs from the bindingpartners present in all other sets of binding partners.

The terms “polypeptide,” “peptide”, and “protein” are usedinterchangeably herein and refer to a polymer of amino acid residues.The terms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to amino acid polymers containing onlynaturally occurring amino acids. The term “binding protein” refers toany protein binding partner other than an antibody, as defined above.

The terms “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid refers to a single-stranded or double stranded polymer andwill generally contain phosphodiester bonds, although in some cases, asoutlined below, nucleic acid analogs are included that may havealternate backbones, comprising, for example, phosphoramide or peptidenucleic acid backbones.

The terms “nucleic acid molecule encoding” “DNA sequence encoding” and“DNA encoding”, as used herein refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

The term, “diagnostic platform”, as used herein, refers to any deviceand/or method that may be used to detect and/or identify a biologicalorganism. For example, a diagnostic platform may be used to detectand/or identify an hMPV viral particle.

The term, “intradermal injection cartridge”, as used herein, refers toany device that is capable of delivering a measured dose of acomposition into the intradermal region of the skin. For example, acartridge may intradermally administer an hMPV MAb such that circulatinghMPV viral particles are detecting by observing the development of aconventional histamine-mediated inflammation reaction (i.e., forexample, a “wheal and flare” reaction).

The term, “porous material”, as used herein, refers to any material thatis permeable to fluids.

The term, “electrical field” as used herein, refers to the relativepositioning of a positive electrode and negative electrode such that anelectrical potential is created between the electrodes. Consequently,any particle within the electrical potential is considered to be “underthe influence of an electric field”.

The term “electric potential”, as used herein, refers to the potentialenergy of a unit positive charge at a point in an electric field that isreckoned as the work which would be required to move the charge to itslocation in the electric field from an arbitrary point having zeropotential (as one at infinite distance from all electric charges) andthat is roughly analogous to the potential energy at a given elevationin a gravitational field.

The term “solid support”, as used herein, refers to any compositionand/or material that is capable of immobilizing a compound including,but not limited to, an antigen (i.e., for example, an hMPV) or anantibody (i.e., for example, an antibody reactive with an hMPV). A solidsupport may include, but is not limited to, beads, strips, wells,microchannels etc.

The term “nitrocellulose test strip”, as used herein, refers to anydevice or composition comprising nitrocellulose wherein a compound maybe detected and/or identified.

The term “ferrofluid”, as used herein, refers to any compositioncomprising iron to which another compound (i.e., for example, anantibody) may be attached. Ferrofluids may also have attachedpurification molecules (i.e., for example, biotin) to facilitate theirremoval and isolation from a biological sample.

An “integrated microfluidic system” is a microfluidic system in which aplurality of fluidic operations are performed. In one embodiment, theresults of a first reaction in the microfluidic system are used toselect reactants or other reagents for a second reaction or assay. Thesystem will typically include a microfluidic substrate, and a fluidicinterface for sampling reactants or other components. A detector and acomputer are often included for detecting reaction products and forrecording, selecting, facilitating and monitoring reactions in themicrofluidic substrate.

A “microfluidic device” is an apparatus or component of an apparatushaving microfluidic reaction channels and/or chambers. Typically, atleast one reaction channel or chamber will have at least onecross-sectional dimension between about 0.1 μm and about 500 μm.

A “reaction channel” is a channel (in any form, including a closedchannel, a capillary, a trench, groove or the like) on or in amicrofluidic substrate (a chip, bed, wafer, laminate, or the like havingmicrofluidic channels) in which two or more components are mixed. Thechannel will have at least one region with a cross sectional dimensionof between about 0.1 μm and about 500 μm.

A “reagent channel” is a channel (in any form, including a closedchannel, a capillary, a trench, groove or the like) on or in amicrofluidic substrate (a chip, bed, wafer, laminate, or the like havingmicrofluidic channels) through which components are transported(typically suspended or dissolved in a fluid). The channel will have atleast one region with a cross sectional dimension of between about 0.1μm and about 500 μm.

A “material transport system” is a system for moving components along orthrough microfluidic channels. Exemplar transport systems includeelectrokinetic, electroosmotic, and electrophoretic systems (e.g.,electrodes in fluidly connected wells having a coupled current and/orvoltage controller), as well as micro-pump and valve systems.

A “fluidic interface” in the context of a microfluidic substrate is acomponent for transporting materials into or out of the substrate. Theinterface can include, e.g., an electropipettor, capillaries, channels,pins, pipettors, sippers or the like for moving fluids into themicrofluidic substrate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents exemplary data showing patterns of IFA staining of MAbsto hMPV on hMPV-infected LLC-MK2 cell cultures. Panels A & B: Granularpatterns (hMPV MAbs from Clone C2C10). Panels C & D: Filamentouspatterns (hMPV MAbs from Clone C2D11). Panels E & F: Foamy patterns(hMPV MAbs from Clone T3H11).

FIGS. 2 (A & B) present exemplary data showing direct fluorescentantibody (DFA) staining of respiratory mucosal cells from two differentNPAs.

FIGS. 2 (C & D) present exemplary data showing hMPV isolation andidentification in LLC-MK2 shell vial cell cultures by using MAbs 48hours after inoculation of two different NPA samples. Panel C: Singleinfected cells. Panel D: A plaque of infected cells with small syncytialformations.

FIG. 3 presents exemplary data showing the typing of reference hMPVstrains in LLC-MK2 cell cultures by type-specific MAbs and indirectimmunofluorescence (IFA) 24 h p.i. Panel A: Type A reference strain I-PV03/01 6621 stained with hMPV Type A MAb. Panel B: Type A referencestrain I-PV 03/01 6621 stained with hMPV Type B MAb. Panel C: Type Breference strain I-PV 03/04 4702 stained with hMPV Type A MAb. Panel D:Type B reference strain I-PV 03/4 4702 stained with hMPV Type B MAb.

FIG. 4 (A-D) presents exemplary data showing the typing of two hMPVisolates recovered in LLC-MK2 cell cultures after an 48 hour incubationby TA and MAbs. Panel A: Type A isolate stained with hMPV Type A MAb;Panel B: Type A isolate stained with hMPV Type B MAb; Panel C: Type Bisolate stained with hMPV Type A MAb. Panel D: Type B isolate stainedwith hMPV Type B MAb.

FIG. 4 (E-H) presents exemplary data showing the typing of hMPV strainsby IFA and type-specific MAbs on respiratory cells from NPA. Panel E:Type A hMPV NPA stained with hMPV Type A MAb; Panel F: Type A hMPV NPAstained with hMPV Type B MAb; Panel G: Type B hMPV NPA stained with hMPVType A MAb; Panel H: Type B hMPV NPA stained with hMPV Type B MAb.

DETAILED DESCRIPTION OF THE INVENTION

This invention is related to the detection and identification ofviruses. In one embodiment, the virus comprises a metapneumovirus. Inone embodiment, the virus infects a mammal. In one embodiment, theinfection may be identified using an antibody. In particular, theantibodies are monoclonal antibodies produced from a hybridoma cellculture.

At least two genetic lineages of hMPV have been identified, with bothlineages circulating during the same season (i.e., for example, Type Aand Type B). The surface glycoproteins, G and SH show the mostdivergence between the two main lineages, with the G protein being moredivergent than observed between human RSV A and B lineages. Unlike othercurrent diagnostic hMPV technologies, the present invention hassuccessfully provided monoclonal antibodies that detect and identify aspecific type of hMPV (i.e., for example, Type A or Type B) and does nothave detectable cross-reactivity with other respiratory viruses or anavian metapneumovirus.

I. Metapneumovirus

Known avian metapneumoviruses belong to four different types (A throughD), with type C being the closest to hMPV. Jacobs et al., “Subtype Bavian metapneumovirus resembles subtype A more closely than subtype C orhuman metapneumovirus with respect to the phosphoprotein, and secondmatrix and small hydrophobic proteins” Virus Res. 92:171-178 (2003); andNjenga et al., “Metapneumovirus in birds and humans” Virus Res.91:163-169 (2003). It cannot be excluded that other,as-yet-unidentified, types of hMPV strains exist that may havesimilarity to avian metapneumovirus types A, B and D. In one embodiment,the present invention contemplates a monoclonal antibody that detectsonly human metapneumovirus epitopes, and without detectablecross-reactivity to other respiratory viruses or the avianmetapneumovirus subtypes.

Some monoclonal antibodies raised to a human metapneumovirus types mayhave significant cross-over reactivity with avian metapneumovirus, orother respiratory viruses. Consequently, it must be empirically shownthat a specific hMPV MAb is at least two-fold more reactive than withother respiratory viruses or the avian metapneumoviruses.

All human metapneumovirus (hMPV) strains recovered until now indifferent countries of the five continents have been classified into twomajor clusters, referred to as types A and B, on the basis of sequencingand phylogenetic analysis of genes L, N, F, or P. Boivin et al.,“Virological features and clinical manifestations associated with humanpneumovirus: A new paramyxovirus responsible for acute respiratory-tractinfections in all age groups” J Infect Dis 186:1330-1334 (2002); Boivinet al., “Global genetic diversity of human metapneumovirus fusion gene”Emerg Infect Dis 10:1154-1157 (2004); Van den Hoogen et al., “A newlydiscovered human pneumovirus isolated from young children withrespiratory tract disease” Nat Med 7:719-724 (2001); and Van den Hoogenet al., “Antigenic and genetic variability of human metapneumoviruses”Emerg Infect Dis 10:658-665 (2004). The recently identified humanmetapneumovirus (hMPV) is the only member of the genus Metapneumovirusthat infects humans (family Paramyxoviridae, subfamily Paramyxovirinae).As discussed above, the genus Metapneumovirus also includes avianpneumoviruses A, B, C, and D.

II. Monoclonal Antibodies

In one embodiment, the present invention contemplates a compositioncomprising a monoclonal antibody raised against a human metapneumovirus(i.e., for example, Type A or Type B human metapneumovirus). In oneembodiment, the monoclonal antibody is at least two-fold more reactivewith human metapneumovirus than with an avian metapneumovirus, or otherrespiratory viruses. In one embodiment, the human metapneumovirusmonoclonal antibody is reactive against type A human metapneumovirus. Inanother embodiment, the human metapneumovirus monoclonal antibody isreactive against type B human metapneumovirus. Although it is notnecessary to understand the mechanism of an invention, it is believedthat the development of monoclonal antibodies having specificity tohuman metapneumovirus are capable of typing all strains previouslycharacterized by sequencing and phylogenetic analysis.

The recent discovery of hMPV as a major respiratory pathogen of infantsand young children has been made possible by means of RT-PCR. Studiesthus far published have mostly been conducted using this molecularapproach. The present invention contemplates hMPV-specific MAbs thatwill now allow the routine use of direct fluorescence antibody (DFA)staining for hMPV detection in NPAs. Advantages of using hMPV-specificMAbs versus an RT-PCT assay include, but are not limited to, quicksample turn-around time and simple laboratory procedures. In addition,MAbs are capable of reacting with all four hMPV subtypes and proves theability of these reagents to detect all known hMPV strains Bastien etal., “Sequence analysis of the N, P, M, and F genes of Canadian humanmetapneumovirus” Virus Res., 93:51-62 (2003); Boivin et al., “Globalgenetic diversity of human metapneumovirus fusion gene” Emerg. Infect.Dis. 10:1154-1157 (2004); Mackay et al., “Use of the P gene to genotypehuman metapneumovirus identifies 4 viral subtypes”, J. Infect. Dis.190:1913-1918 (2004); and van den Hoogen et al., “Antigenic and geneticvariability of human metapneumovirus” Emerg. Infect. Dis. 10:658-665(2004). This simple diagnostic approach cannot be attained using RT-PCR,which requires a specific and technically difficult procedure for eachdiagnostic test.

Development of monoclonal antibodies having specificity for a humanmetapneumovirus (hMPV MAbs) is an important advance in the field ofrapid direct diagnosis of respiratory tract viral infections. Followingthe introduction of hybridoma technology, MAbs to other respiratoryviruses were developed and made commercially available. MAbs to hMPV,however, were not available.

Since then, DFA staining using MAbs has become the most rapid techniquefor direct diagnosis of acute respiratory infections, taking only 2 to 3hours to perform. Expertise in reading the results of DFA assays andgood-quality smears of respiratory cells are preferred for reliableperformance of the DFA assay. In parallel, molecular assays aimed atamplifying viral genomes directly in clinical samples have beendeveloped and compared to DFA staining for diagnosis of respiratoryviral infections (i.e., for example, RT-PCR). Rovida et al., “MonoclonalAntibodies versus reverse transcription-PCR for detection of respiratoryviruses in a patient population with respiratory tract infectionsadmitted to hospital” J Med Virol. 75:336-347 (2005). These comparisons,however, did not include hMPV MAbs because of their commercialunavailability.

Initially developed RT-PCR protocols were unable to detect type Bstrains in clinical samples. van de Hoogen et al., “Prevalence andclinical symptoms of human metapneumovirus infection in hospitalizedpatients” J. Infect. Dis. 188:1571-1577 (2003). This deficiency wascorrected by the identification of a second primer that is capable ofidentifying type B strains. Sararini et al., “Detection andpathogenicity of human metapneumovirus respiratory infection inpediatric Italian patients during a winter-spring season” J Clin Virol35: 59-68 (2006). Consequently, it is necessary to perform RT-PCRprotocols using both sets of primer pairs to detect all hMPV typesand/or subtypes. Although it is not necessary to understand themechanism of an invention, it is believed that type-specific hMPV MAbsas contemplated by the present invention perform better than RT-PCRmethods when the RT-PCR primers are not carefully selected based uponconserved regions of the genome.

Further, available research regarding hMPV MAb development has notreported any ability to differentiate between hMPV Type A viral antigensand hMPV Type B viral antigens. Landry et al., “Detection of humanmetapneumovirus in clinical samples by immunofluorescence staining ofshell vial centrifugation cultures prepared from three different celllines” J Clin Microbiol. 43:1950-1952 (2005); and De Jong et al., “Viruscausing respiratory tract illness in susceptible mammals” United StatesPatent Application Publ. No. 2005/0053919 (2005). Specifically, De Jonget al. contemplates using antibodies that have sufficientcross-reactivity between human metapneumovirus and avian metapneumovirussuch that an antibody raised to a human metapneumovirus may be used todetect an avian metapneumovirus and vice versa. Clearly, these hMPV MAbshave limited and/or no use for clinical diagnosis and treatment.

In one embodiment, the present invention contemplates hMPV MAbscomprising a wide reactivity with all known hMPV subtypes. In oneembodiment, the hMPV MAb has selectivity for hMPV Subtype A. In anotherembodiment, the hMPV MAb has selectivity for hPMV Subtype B. In oneembodiment, an hMPV MAb selectively detects an hMPV antigen using anasopharengeal aspirate (NPA) smear. In another embodiment, an hMPV MAbselectively detects an hMPV antigen using an LLC-MK2 shell vial cellcultures inoculated with NPAs.

In one embodiment, Type A and Type B hMPV strains (1-PV 03/01 6621 andI-PV 03/04 4702, respectively) were propagated in LLC-MK2 cell cultures.Gerna et al., “Changing circulation rate of human metapneumovirusstrains and types among hospitalized pediatric patients during threeconsecutive winter-spring seasons” Arch Virol 150:2365-2375 (2005). Inanother embodiment, virus suspensions were first clarified and thenconcentrated by ultracentrifugation. Although it is not necessary tounderstand the mechanism of an invention, it is believed that theclarification and ultracentrifugation steps results in an hMPV MAbpreparation that has little or no non-specific binding.

Then, BALB/C mice were inoculated according to standard procedures.Percivalle et al., “Rapid detection of human metapneumovirus strains innasopharyngeal aspirates and shell vial cultures by monoclonalantibodies” J Clin Microbiol 43:3443-3446 (2005). Following fusion ofmouse spleen cell suspensions with Sp2/0Ag14 myeloma cells, the MAbsexpressed by the hybridomas were tested for specific reactivity withhMPV by enzyme-linked immunosorbent assay and the indirect fluorescentantibody (IFA) assay. Following cloning and subcloning, MAbs previouslyselected for specific reactivity with hMPV were tested for typespecificity by IFA using these Type A and Type B hMPV strains.

hMPV MAb specific reactivity to hMPV proteins was determined usingWestern blots of sucrose-purified reference hMPV strains CAN83 (Type A)and CAN75 (Type B). These reference strains are reported in: Peret etal., “Characterization of human metapneumoviruses isolated from patientsin North America” J Infect Dis 185:1660-1663 (2002). Further, hMPV MAbswere tested by IFA on LLC-MK2 cells, which were infected withrecombinant human parainfluenza virus type 1 expressing the hMPV fusion(F), small hydrophobic (SH), and the attachment glycoprotein (G) ofeither CAN83 (Type A) and CAN75 (Type B). Newman et al., “Sequenceanalysis of the Washington/1964 strain of human parainfluenza virus type1 (HPIV1) and recovery and characterization of wild-type recombinantHPIV1 produced by reverse genetics” Virus Genes 24:77-92 (2002); andSkiadopoulos et al., “The two major human metapneumovirus geneticlineages are highly related antigenically, and the fusion (F) protein isa major contributor to this antigenic relatedness” J Virol 78:6927-6937(2004). Both type-specific MAbs were found to react with the F proteinof the same virus type by both IFA and Western blot (i.e., an hMPV TypeA MAb reacts with an hMPV Type A F protein and an hMPV Type B MAb reactswith an hMPV Type B F protein).

Two hMPV MAbs (i.e., for example, clones F4A1 (IgG1) and CB7F3 (IgG1)were reactive by both DFA and ELISA assays with either type A or type BhMPV strains. In one embodiment, clone F4A1 is selective for hMPVsubtypes A1 and A2. In one embodiment, clone CB7F3 is selective for hMPVsubtypes B1 and B2. Gerna et al., “Simultaneous detection and typing ofhuman metapneumovirus strains in nasopharyengeal secretions and cellcultures by monoclonal antibodies. J Clin Virol 35:113-115 (2006) ThehMPV MAbs were further tested for cross-reactivities with conventionalrespiratory viruses (i.e., for example, influenza viruses A and B,parainfluenza virus types 1-4, human respiratory syncytial virus, humanadenovirus, human coronaviruses 229, OC43 and NL63, and rhinoviruses).No cross-reactivity with any known respiratory viruses was detected foreither one of the two selected MAbs. Further, these hMPV MAbs showlittle or no non-specific staining (i.e., background interference). hMPVMAbs created according to the above described protocol, were tested byDFA using 67 NPA samples. Specifically, 24 hMPV strains were typed byhMPV MAbs using duplicate NPA slides. The data showed that sixteen (16)strains were found to belong to Type A, and eight (8) strains were foundto belong to Type B. See Table I. These results exactly matched thoseobtained by sequencing and phylogenetic analysis. Gerna et al., supra.

In parallel, MAbs specific for Type A or Type B human metapneumoviruswere also used to test eighteen (18) NPA samples positive for differentrespiratory viruses. The data clearly show that no cross-reactivity withany of the other respiratory viruses tested was found, therebydemonstrating that hMPV MAbs as contemplated by the present inventionhave a 100% specificity for hMPV. In addition, hMPV type-specific MAbswere tested against respiratory cells from twenty-five (25) NPA samplesnegative for respiratory viruses. Again, no non-specific reactivity withuninfected respiratory cells was detected. See Table I.

TABLE I hMPV typing of 24 hMPV-positive NPA samples by DFA using MAbsspecific for either Type A or Type B hMPV compared to typing byphylogenetic analysis Typing by hMPV MAbs Respiratory virus No. NPAstested Type A Type B hMPV type A^(a) 16 16 0 hMPV type B^(a) 8 0 8Influenza virus A 2 0 0 Influenza virus B 2 0 0 Parainfluenza virus 1-33 0 0 Respiratory syncytial virus 3 0 0 Adenovirus 2 0 0 Humancoronaviruses 3 0 0 Rhinoviruses 3 0 0 None (cells from NPA) 25 0 0 Inaddition, 18 NPAs positive for different respiratory viruses and 25 NPAsamples negative for respiratory viruses were tested as controls. ^(a)Astyped by sequencing and phylogenetic analysis.

Furthermore, some NPA samples positive for hMPV using RT-PCR werere-typed using MAbs having specificity for human metapneumovirus ascontemplated by the present invention using LLC-MK2 cell cultures (i.e.,for example, by using a shell vial centrifugation technique). While12/12 (100%) of samples inoculated as fresh NPAs were recovered in cellcultures and typed, only 8/25 (32%) samples thawed once or twice, couldbe typed. Thus, both hMPV recovery and typing are optimally achieved byinoculating fresh samples onto cell cultures.

Morphological patterns of the two type-specific MAbs in cell culturesinfected with reference strains are shown. See FIG. 3. In addition, IFApatterns observed in respiratory tract cells from hMPV-infected NPAsamples, as well as in LLC-MK2 cell cultures following hMPV isolation,are shown for both Type A and Type B hMPV strains. FIGS. 4 (A-D) andFIGS. 4 (E-H), respectively. The staining intensity ranged from 1+ to 4+in different cells, while the staining pattern was similar to thegranular pattern of one of three MAbs included in the pool for hMPVdetection. Percivalle et al., “Rapid detection of human metapneumovirusstrains in nasopharyngeal aspirates and shell vial cultures bymonoclonal antibodies” J Clin Microbiol 43:3443-3446 (2005).

Although it is not necessary to understand the mechanism of aninvention, it is believed that the development of MAbs havingspecificity to hMPV are capable of classifying all hMPV strains testedinto Type A or Type B, thereby exactly matching results given bysequencing and phylogenetic analysis. It is further believed that sinceboth Type A and Type B hMPV have been found to circulate at differentrates in different years hMPV typing, the hMPV MAb's contemplated hereinmay be useful for epidemiological research and prediction. Gerna et al.,“Changing circulation rate of human metapneumovirus strains and typesamong hospitalized pediatric patients during three consecutivewinter-spring seasons” Arch Virol 150:2365-2375 (2005).

hMPV typing by MAbs is highly preferable over typing by phylogeneticanalysis in terms of practicality, rapidity and cost-effect benefits.Given the 100% sensitivity and specificity of type-specific MAbs withrespect to hMPV detection by the MAb pool, detection and typing of newhMPV strains by MAbs may be performed simultaneously in viral diagnosticlaboratories. Percivalle et al., “Rapid detection of humanmetapneumovirus strains in nasopharyngeal aspirates and shell vialcultures by monoclonal antibodies” J Clin Microbiol 43:3443-3446 (2005);and Gerna et al., supra.

III. Diagnostics

Some embodiments of the present invention contemplate direct antibodydetection and identification of hMPV virus proteins that represent animprovement of immunological identification of hMPV strains was achievedby direct fluorescent antibody (DFA) staining of cells from NPA samples.Ebihara et al., “Detection of human metapneumovirus antigens innasopharyngeal secretions by an immunofluorescent-antibody test” J ClinMicrobiol 43:1138-1141 (2005).

A wide spectrum of clinical symptoms is associated with hMPV infection.In the general community, hMPV-infected adults mainly suffer from upperrespiratory tract illnesses (common cold-like symptoms) such as cough,rhinorrhea, hoarseness, sore throat and sometimes fever. In hospitalizedchildren, patients with underlying disease, immunocompromisedindividuals, and fragile elderly, hMPV disease may be more severe,involving the lower respiratory tract. Clinical diagnoses may range fromrhinopharyngitis to bronchitis and pneumonia, and some patients may beadmitted to intensive care units. In addition, diarrhea, vomiting, rash,febrile seizures, feeding difficulties, conjunctivitis and otitis mediahave all been reported. The wide spectrum of hMPV-induced illnessesreported thus far are similar to those caused by RSV and influenza virusinfections.

Some embodiments of the present invention contemplate the administrationof monoclonal antibodies having specificity for a human metapneumovirus.In one embodiment, the monoclonal antibody administration is given to asubject such that an hMPV infection is prevented (i.e., prophylacticadministration). In one embodiment, the monoclonal antibodyadministration is given to a subject such that an existing hMPVinfection is reduced, ameliorated, and/or eliminated.

MAb detection of hMPV antigens was reported using inoculated cellcultures. Percivalle et al., “Rapid detection of human metapneumovirusstrains in nasopharyngeal aspirates and shell vial cultures bymonoclonal antibodies” J Clin Microbiol 43:3443-3446 (2005). Althoughsensitivity and negative predictive value of MAbs were somewhat lowerthan those achieved by RT-PCR, rapidity of turnaround time, simplicityof test performance, and MAb specificity provide advantages that favorthis immunological approach over conventional detection techniques.

An hMPV MAb raised to a matrix protein (MAb-8; MAB8510, ChemiconInternational, Temecula, Calif.) was reported as capable of detectinghMPV in shell vial centrifugation cultures. Landry et al., “Detection ofhuman metapneumovirus in clinical samples by immunofluorescence stainingof shell vial centrifugation cultures prepared from three different celllines” J Clin Microbiol 43:1950-1952 (2005). MAb-8 was unable to detecthMPV in clinical samples, however, because of uncontrollablenon-specific staining (i.e., for example, resulting from backgroundcellular material and/or other viruses). This nonspecific backgroundstaining made reading very tedious and interpretation was difficult.Specific recommendations for using MAb-8 included experience indistinguishing specific versus nonspecific staining and to alwaysinclude both positive and negative controls.

Rapid immunological diagnosis of conventional respiratory virusinfections is currently performed by direct fluorescent antibody (DFA)staining of respiratory cells present in nasopharyngeal aspirate (NPA)samples using virus-specific monoclonal antibodies (MAbs). Rovida etal., “Monoclonal Antibodies versus reverse transcription-PCR fordetection of respiratory viruses in a patient population withrespiratory tract infections admitted to hospital” J Med. Virol.75:336-347 (2005). Therefore, MAbs with specific reactivity to hMPVantiens within biological samples (i.e., for example, NPAs) are needed,although they are not yet commercially available. This inventiondiscloses MAbs specific for Type A hMPV and Type B hMPV and their usefor DFA staining of NPA samples and virus identification in shell vialcell cultures.

A. Diagnostic Platforms

Some embodiments of the present invention can provide a high-throughput,simple, and rapid diagnostic clinical tool having many advantages overRT-PCR protocols. For instance, the use of hMPV MAbs can be incorporatedinto many types of immunological assay devices and techniques (i.e., forexample, diagnostic platforms) that are already commercially available.

1. Cartridges

In one embodiment, the present invention contemplates a diagnosticplatform comprising an intradermal injection of an hMPV MAb fordiagnostic testing. Similar to determining the immunity status of theanimal against tuberculosis and the immediate hypersensitivity status ofType I allergic diseases, a prefilled container administers hMPV MAbinto a subject either having, or at risk for, an hMPV infection. Apositive reaction is then noted upon redness, swelling, or tenderness atthe injection site indicating that the MAb injection initiated aninflammatory reaction due to the presence of hMPV antigens within thesubject.

An intradermal injection is made by delivering the substance into theepidermis and upper layer of the dermis. Below the dermis layer issubcutaneous tissue (also sometimes referred to as the hypodermis layer)and muscle tissue, in that order. There is considerable variation in theskin thickness both between individuals and within the same individualat different sites of the body. Generally, the outer skin layer,epidermis, has a thickness between 500-200 microns, and the dermis, theinner and thicker layer of the skin, has a thickness between 1.5-3.5 mm.Therefore, a needle cannula that penetrates the skin deeper than about3.0 mm has a potential of passing through the dermis layer of the skinand making the injection into the subcutaneous region, which may resultin an insufficient immune response, especially where the substance to bedelivered intradermally has not been indicated for subcutaneousinjection. Also, the needle cannula may penetrate the skin at tooshallow a depth to deliver the substance and result in a “wet injection”because of reflux of the substance from the injection site.

For example, an intradermal needle assembly compatible with aprefillable container having a reservoir capable of storing hMPV MAb forinjection into the skin of an animal includes a hub portion beingattachable to the prefillable container storing the substance, a needlecannula supported by the hub portion and having a forward tip extendingaway from the hub portion, and a limiter portion surrounding the needlecannula and extending away from the hub portion toward the forward tipof the needle cannula, the limiter including a generally flat skinengaging surface extending in a plane generally perpendicular to an axisof the needle cannula and adapted to be received against the skin of theanimal to administer an intradermal injection of the substance, theneedle forward tip extending beyond the skin engaging surface a distanceapproximately 0.5 mm to 3.0 mm wherein the limiter portion limitspenetration of the needle into the dermis layer of skin of the animal sothat the vaccine is injected into the dermis layer of the animal. Alchaset al., “Intradermal needle” U.S. Pat. No. 6,843,781 (2005). (hereinincorporated by reference).

2. Cell Based Assays

hMPV may be detected using a diagnostic platform comprising cell culture(i.e., for example, tMK cells (RIVM, Bilthoven, The Netherlands)technique using a plurality of well plates containing glass slides(Costar, Cambridge, UK). The cells are incubated with a medium asdescribed below supplemented with 10% fetal bovine serum (BioWhittaker,Vervier, Belgium). Before hMPV inoculation, the plates were washed withPBS and supplied with Eagle's MEM with Hanks' salt (ICN, Costa mesa,Calif.) supplemented with 0.52/liter gram NaHCO₃, 0.025 M Hepes(Biowhittaker), 2 mM Iglutamine (Biowhittaker), 200 units/literpenicilline, 200 μg/liter streptomycin (Biowhittaker), 1 gram/literlactalbumin (Sigma-Aldrich, Zwindrecht, The Netherlands), 2.0 gram/literD-glucose (Merck, Amsterdam, The Netherlands), 10 gram/liter peptone(Oxoid, Haarlem, The Netherlands) and 0.02% trypsin (Life Technologies,Bethesda, Md.).

Plate inoculation may be performed with a supernatant of thenasopharyngeal aspirate samples (i.e., for example, 0.2 ml per well intriplicate) followed by centrifuging at 840×g for one hour. Afterinoculation, the plates are incubated at 37° C. for a maximum of 14 dayschanging the medium once a week and cultures were checked daily forcytopathic effects (CPE). After 14 days, cells can be scraped from asecond passage and incubated for another 14 days. This step may berepeated for a third passage. The glass slides were used to demonstratethe presence of the virus by indirect IFA.

CPE is generally observed after a third passage, at day 8 to 14,depending on the isolate. CPE induced by hMPV is virtuallyindistinguishable from that caused by hRSV or hPIV in tMK or other cellcultures. However, hRSV induces CPE starting around day 4. CPE may becharacterized by syncytia formation, after which the cells showed rapidinternal disruption, followed by detachment of cells from the monolayer.If CPE is difficult to observe, IFA may be used to confirm the presenceof the virus in these cultures. De Jong et al., “Virus causingrespiratory tract illness in susceptible mammals” United States PatentApplication 2005/0053919 (2005)(herein incorporated by reference).

3. Porous Media Support Material

In one embodiment, an hMPV can be retained on a diagnostic platformcomprising a porous media under the influence of an electrical fieldwith fluid flow. In this manner, an hMPV can be concentrated on theporous media with a low electrical field with fluid flow. In oneembodiment, the virus can then be removed from the porous media byreducing or shutting off the electrical field. Although it is notnecessary to understand the mechanism of an invention, it is believedthat unique sets of conditions (i.e., solution flow rate and electricalfield strength) are capable of immobilizing a virus using a given mediaand solution composition.

For example, elution electrophoresis uses a flow-through porous mediawith an applied electrical field. The electrical field causes theselective retention of compounds (i.e., for example, viral proteinand/or nucleic acids). The retention of these compounds is dependent onthe flow rate (velocity) and electrical field strength in a given porousmedia. The chemical and physical nature of the porous media plays a rolein the separation process. Very selective separations are possible usingthis technology because the electrical field strength, flow velocity,and porous media can be varied for each separation. Individualcomponents in the mixture can be retained to varying degrees by theseparation conditions used. The degree of retention is determined by thechemical and physical properties of each component. Cole K. D.,“Concentration and size-fractionation of nucleic acids and viruses inporous media” U.S. Pat. No. 5,707,850 (1998)(herein incorporated byreference).

The flow rate of the solution through the porous media is also avariable in this process. The flow rate can be held constant or varied.Flow rate can be changed in steps or continuously (a gradient) duringthe separation process.

In one embodiment, the media comprises a porous and fairly neutralmaterial and includes, but is not limited to, Sephadex®, dextran,cellulose, acrylamide, polymeric particles, or silica. In oneembodiment, the material comprises beads and may be applied with acolumn. In another embodiment, the material is applied on membranes,and/or thin layers.

The pH, ionic strength, and composition of the solution can also bemodified during the separation process. The pH of the solutiondetermines the charge and electrophoretic mobility of each component. Itis possible to lower the pH significantly, so that the proteins willbecome positively charged and not be retained, while nucleic acids willstill be negatively charged and retained on the porous media. The ionicstrength and ionic composition of the solution will influence theelectrical field strength and influence interactions of components withthe porous media. The solution can be varied in its composition toinclude additives that vary the interactions of the components with theporous media (such as detergents and zwitterions). Modification of thecomposition of the solution during the separation is therefore avariable that can be used to achieve selective separations.

The electrical field strength and polarity can also be changed duringthe separation (i.e., for example, a negative field or a positivefield). The field can be changed continuously or in a step-wise fashion.Alternating and pulsed electrical fields can also be used. Any directionof pulsing may be used, including reverse flow, right angles,orthogonal, etc. Although it is not necessary to understand themechanism of an invention, it is believed that low electrical fieldsused to separate the DNA are advantageous because they do not result incolumn heating which can lead to temperature gradients that ruinresolution and can denature delicate biological molecules. Further, lowelectrical fields have very little effect on the retention of proteinsso that they pass through the column.

Recovery of products can be automated, thereby making the processcommercially viable. The methods can be used for protein recovery,removal of unwanted proteins, and/or isolation of viruses (i.e., forexample, human metapneumovirus). The process can also be used toseparate nucleic acids from proteins and/or viruses from proteins.

Elution electrophoretic separation includes, but is not limited to, azone of porous media, electrodes for applying an electrical field acrossthe media, a means of flowing solution through the porous media (such asa pump), a device to introduce sample to be separated (i.e., forexample, an automated microsyringe), and a device to collect thesolution.

For example, one elution electrophoresis system utilizes a porous packedbed providing protein separation and a flow photometer or colorimeterfor continuous monitoring of the eluate. In one embodiment, an elutionelectrophoresis system is capable of rapid, high-resolution analysis ofhMPV proteins such that they may be detected by a variety ofpost-separation methods. For example, such a system may comprise acooled separation column (i.e., for example, 3 mm in diameter and 40 cmlong) containing polyacrylamide beads. hMPV protein samples areintroduced, via a microsyringe, through a septum at the column midpoint.Typical analyses requires an electrophoresis time of about 30 min at1200 V. Scott et al., “Automated elution electrophoresis: a potentialclinical tool” Clin Chem. 1975 21(9):1217-20 (1975).

4. Dipsticks and Other Solid Supports

Another diagnostic platform for the identification of hMPV types andsubtypes comprises devices and materials enabling the detection of hMPVin biological materials, and is particularly suited for screening largeamounts of samples. One embodiment comprises one or more hMPV-specificmonoclonal antibodies and a solid support. In one embodiment, the solidsupport comprises a microtiter plate, wherein the microtiter plate isoptionally coated with the monoclonal antibodies. In another embodiment,the solid support comprises a test strip, wherein the test strip isoptionally coated with the monoclonal antibodies. In one embodiment, thetest strip comprises nitrocellulose. In one embodiment, a secondaryanti-mouse antibody that is coupled with an enzyme and its substrate orany other molecular compound for a detection reaction (i.e., forexample, a peroxidase-labeled anti-mouse IgG antibody, TMB or any otherperoxidase substrate).

In another embodiment, the method provides a dipstick format and iswithout need of radioactive tracers, enzymes or substrates and mayrequire no more than a single step. This one-step procedure involves thecapture of an hMPV viral protein with one of the hMPV-specific MAbswhich are immobilized on a solid support (i.e., for example, anitrocellulose test strip). These captured hMPV viral proteins may thenbe detected directly by a second antibody. In one embodiment, a detectorcomplex results in the formation of colored spots on the test stripwhich are visible in less than 30 minutes depending on the concentrationof the test sample. The spots are a permanent record of the test resultand, longer exposures increase the sensitivity of the test withoutgenerating higher background. Korth et al., “Immunological detection ofprions” U.S. Pat. No. 6,765,088 (2004)(herein incorporated byreference).

The biological material containing the hMPV sample can be insoluble orsoluble in buffer or body fluids. The biological material can be derivedfrom any part of the body (i.e., for example, from the brain, or tissuesections). Homogenates may be prepared or any body fluid (i.e., forexample, cerebrospinal fluid, urine, saliva, or blood). In the case ofbody fluids, fluid-resident cells (i.e., for example, white blood cells)can be purified and analyzed either in immunohistochemistry or as ahomogenate.

5. Microfluidics

Some embodiments of the present invention contemplate a diagnosticplatform comprising a high-throughput microfluidic device and/or methodcomprising hMPV MAbs. In one embodiment, the device is capable ofcontacting hMPV MAbs with hMPV viral particles, such that the viralparticles are detected, and identified within the microfluidic device

Although it is not necessary to understand the mechanism of aninvention, it is believed that suitable microfluidic substrate materialsare generally selected based upon their compatibility with theconditions present in the particular operation to be performed by thedevice. For example, such conditions can include, but are not limitedto, extremes of pH, temperature, salt concentration, and application ofelectrical fields. Additionally, substrate materials are also selectedfor their inertness to critical components of an analysis or synthesisto be carried out by the device.

In one embodiment, a substrate material may be selected from the groupcomprising glass, quartz, silicon, or other polymeric substrates (i.e.,for example, plastics). In the case of conductive or semi-conductivesubstrates, it is occasionally desirable to include an insulating layeron the substrate. This is preferable when the device incorporateselectrical elements, e.g., electrical fluid direction systems, sensorsand the like. In the case of polymeric substrates, the substratematerials may include, but are not limited to, rigid, semi-rigid,non-rigid, opaque, semi-opaque or transparent, depending upon the usefor which they are intended. For example, devices which include, forexample, an optical, spectrographic, photographic or visual detectionelement, will generally be fabricated, at least in part, fromtransparent materials to allow, or at least, facilitate that detection.Alternatively, transparent windows of, e.g., glass or quartz, areoptionally incorporated into the device for these types of detectionelements. Additionally, the polymeric materials optionally have linearor branched backbones, and can be crosslinked or non-crosslinked.Examples of polymeric materials include, but are not limited to,polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC)polystyrene, polysulfone, polycarbonate, or polymethylmethacrylate(PMMA).

In certain embodiments, the microfluidic substrate comprises one or moremicrochannels for flowing reactants and products. In one embodiment, atleast one of these channels typically has a very small cross sectionaldimension, e.g., in the range of from about 0.1 μm to about 500 μm. Inone embodiment, a cross-sectional dimension of the channels may range offrom about 1 μm to about 200 μm, preferably in the range of from about0.1 μm to about 100 μm, but more preferably in the range of about 1 μmto 100 μm. In order to maximize the use of space on a substrate,alternative geometries, including but not limited to serpentine, sawtooth or other channel geometries, may be used. Although it is notnecessary to understand the mechanism of an invention, it is believedthat these alternative geometries facilitate separation of reactionproducts or reactants and increase the channel length within a definedsurface area. Substrates may be of essentially any size, with areatypical dimensions of about 1 cm² to 10 cm².

Some embodiments of the present invention contemplate a microfluidicdevice comprising one or more chambers, channels or the like, whereinthe chambers and/or channels are fluidly connected to allow transport offluid among the chambers and/or channels. By “microfluidic” is generallymeant fluid systems, e.g., channels, chambers and the like, typicallyfabricated into a solid typically planar substrate, and wherein thesefluid elements have at least one cross-sectional dimension in the rangeof from about 0.1 to about 500 μm. Typically, the cross sectionaldimensions of the fluid elements will range from about 1 μm to about 200μm. A “chamber” will typically, though not necessarily, have a greatervolume than a channel, typically resulting from an increasedcross-section having at least one dimension from about 10 to about 500μm, although, as for channels, the range can span, e.g., 0.1 to about500 μm. Although generally described in terms of channels and chambers,it will generally be understood that these structural elements areinterchangeable, and the terms are used primarily for ease ofdiscussion. By “fluidly connected”, or “in fluid communication”, or “inliquid communication” is meant a junction between two regions, e.g.,chambers, channels, wells etc., through which fluid freely passes. Suchjunctions may include ports or channels, which can be clear, i.e.,unobstructed, or can optionally include valves, filters, and the like,provided that fluid freely passes through the junction when desired.

In one embodiment, the present invention contemplates a method providinga microfluidic device that is used for separating biological particles(i.e., for example, viral particles or antibodies). In one embodiment, avirus membrane protein (i.e., for example, an hMPV membrane protein) maybe complexed with a capture bead within a reaction channel. In oneembodiment, diagnostic reagents (i.e., for example, an hMPV monoclonalantibody) may be stored in a reagent well. Other solutions, such asbuffers for material transport, and or reagents may be stored in otherwells. In one embodiment, an electropipettor channel is fluidlyconnected to the reaction channel thereby providing an automatedhigh-throughput diagnostic system.

In some embodiments, the viral proteins may be attached to capture beads(i.e., for example, posts, magnetic beads, polymer beads or the like)and electrokinetically transported to a bead capture area. In oneembodiment, an electropipettor channel provides an appropriately labeledmonoclonal antibodies that is also transported to the bead capture area.In one embodiment, the labeled monoclonal antibody attaches to the viralproteins thereby allowing detection and identification of the virus whenthe antibody/viral protein complex is eluted. Optionally, theantibody/viral protein complex may be washed from the bead capture areausing a loading buffer and optionally electrophoresed through a sizeseparation microchannel. Knapp et al., “Microfluidic devices and methodsfor separation” U.S. Pat. No. 6,444,461 (2002)(herein incorporated byreference).

Alternatively, hMPV may be captured using isolation fluids such as thosedesigned to be used with the CellTracks® AutoPrep System (Immunicon,Huntingdon, Pa.). Owen et al., “Magnetic-polymer particles” U.S. Pat.No. 4,795,698 (1989) (herein incorporated by reference). For example, aferrofluid comprising a magnetic core coated with BSA, can be conjugatedwith hMPV MAbs for capturing hMPVs. Ferrofluid particles are colloidal,which permit long incubations without ferrofluid settling within areaction chamber. In one embodiment, the present invention contemplatesa ferrofluid comprising an hMPV-specific MAb attached to a particlecomprising an oxide and magnetite, having an average diameter ofapproximately 145 nm. In one embodiment, greater than 85% of the oxideis Fe₃O₄. In one embodiment, the magnetite is approximately 80% (w/w).In one embodiment, the particle comprises a magnetic susceptibility ofapproximately 125 emu/gm, wherein there are approximately 4×10¹¹particles/mg lion, and further wherein each particle comprisesapproximately 3 fg Fe₃O₄ and 1 fg BSA. In one embodiment, the hMPV MAbferrofluid may further comprise biotinylated molecules. In oneembodiment, the ferrofluid comprises approximately 15,000 smallbiotinylated molecules/particle. In another embodiment, the ferrofluidcomprises approximately 5,000 large biotinylated molecules/particle. Inyet another embodiment, the ferrofluid comprises approximately 50-150 μgmonobiotinylated MAb/mg Iron. In still yet another embodiment, theferrofluid comprises approximately 10 nanomoles biotin-FITC/mg Iron.

Many microfabrication techniques may be used to manufacture thesemicroscale elements onto the surface of a substrate. For example,lithographic techniques are generally employed in fabricating microscaledevices using substrates including, but not limited to, glass, quartz orsilicon. Lithographic techniques are common in the semiconductormanufacturing industries such as photolithographic etching, plasmaetching or wet chemical etching. Ghandi, S. K., In: VLSI Principles:Silicon and Gallium Arsenide, NY, Wiley (see, esp. Chapter 10).Alternatively, micromachining methods such as laser drilling, airabrasion, micromilling and the like may be employed. Polymericsubstrates may be manufactured by techniques including, but not limitedto, injection molding or stamp molding methods. Large numbers ofpolymeric substrates may be produced using: i) rolling stamps producinga large sheet of microscale substrate; or ii) polymer microcastingtechniques where the substrate is polymerized within a micromachinedmold. Parce et al., “Controlled fluid transport in microfabricatedpolymeric substrates” U.S. Pat. No. 5,885,470 (1999) (hereinincorporated by reference).

IV. Preparation of Binding Partners

Some embodiments of the present invention contemplate the binding ofmonoclonal antibodies with binding partners (i.e., for example, viralparticles) for detection and identification. In one embodiment, theviral particles may include, but are not limited to, nucleic acidsand/or proteins. In one embodiment, an hMPV MAb may become a bindingpartner with an hMPV viral particle.

A. Nucleic Acids

Nucleic acids for use as binding partners in this invention can beproduced or isolated according to any of a number of methods. In oneembodiment, the nucleic acid can be an isolated naturally occurringnucleic acid (e.g., genomic DNA, cDNA, mRNA, etc.). Methods of isolatingnaturally occurring nucleic acids have been reported. (see, e.g.,Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd Ed.),Vol. 1 3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Nucleic acids useful in the invention can also be amplified from anucleic acid sample (i.e., for example, an hMPV nucleic acid sample). Anumber of amplification techniques have been described, but thepolymerase chain reaction (PCR) is the most widely used. U.S. Pat. Nos.4,683,202, 4,683,195, 4,800,159, and 4,965,188 (all of which are hereinincorporated by reference). Briefly, PCR entails hybridizing two primersto substantially complementary sequences that flank a target sequence ina nucleic acid. A repetitive series of reaction steps involving templatedenaturation, primer annealing, and extension of the annealed primers bya DNA polymerase results in the geometric accumulation of the targetsequence, whose termini are defined by the 5′ ends of the primers. Asdenaturation is typically carried out at temperatures that denature mostDNA polymerases (e.g., about 93° C.-95° C.), a thermostable polymerase,such as those derived from Thermus thermophilus, Thermus aquaticus(Taq), or Thermus flavus, is typically used for extension to avoid theneed to add additional polymerase for each extension cycle.

In one embodiment, the nucleic acid is created de novo, e.g., throughchemical synthesis. In one embodiment, nucleic acids (e.g.,oligonucleotides) are chemically synthesized according to the solidphase phosphoramidite triester method. Beaucage et al., TetrahedronLetts. 22:1859-1862 (1981). In one embodiment, the nucleic acid issynthesized using an automated synthesizer. Needham-VanDevanter et al.,Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides, where necessary, is typically performed by eithernative acrylamide gel electrophoresis or by anion-exchange HPLC. Pearsonet al., J. Chrom. 255:137-149 (1983). The sequence of the syntheticoligonucleotides can be verified using chemical degradation. Maxam etal., Meth. Enzymol. 65:499-560 (1980).

B. Antibodies/Antibody Fragments

In one embodiment, antibodies or antibody fragments for use as bindingpartners can be produced by a number of methods. Harlow & Lane, In:Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988);and Asai, “Antibodies in Cell Biology”, In: Methods in Cell Biology Vol.37: Academic Press, Inc. N.Y (1993). In one embodiment, antibodies areproduced by immunizing an animal (e.g. a rabbit) with an immunogencontaining the epitope to be detected. A number of immunogens may beused to produce specifically reactive antibodies. Recombinant proteinsare the preferred immunogens for the production of the correspondingmonoclonal or polyclonal antibodies. Naturally occurring protein mayalso be used either in pure or impure form. Synthetic peptides are alsosuitable and can be made using standard peptide synthesis chemistry.Barany et al., “Solid-Phase Peptide Synthesis”; pp. 3-284 In: ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield et al., J. Am. Chem. Soc.,85:2149-2156 (1963); and Stewart et al., In: Solid Phase PeptideSynthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984).

Many methods of production of polyclonal antibodies have been previouslyreported. In brief, an immunogen is mixed with an adjuvant and an animalis immunized. The animal's immune response to the immunogen preparationis monitored by taking test bleeds and determining the titer ofreactivity to the immunogen. When appropriately high titers of antibodyto the immunogen are obtained, blood is collected from the animal and anantiserum is prepared. If desired, the antiserum can be furtherfractionated to enrich for antibodies having the desired reactivity.(See Harlow et al., supra).

Many methods of production of monoclonal antibodies have also beenreported. Briefly, spleen cells from an animal immunized with a desiredantigen are immortalized, commonly by fusion with a myeloma cell. Kohleret al., Eur. J. Immunol. 6:511-519 (1976). Alternative methods ofimmortalization include, but are not limited to, transformation withEpstein Barr Virus, oncogenes, or retroviruses. Colonies arising fromsingle immortalized cells are screened for production of antibodies ofthe desired specificity and affinity for the antigen, and yields of themonoclonal antibodies produced by such cells can be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, DNA sequences encoding a monoclonalantibody or a binding fragment thereof can be isolated by screening aDNA library from human B cells. Huse et al., Science, 246:1275 1281(1989). Such isolated sequences can then be expressed recombinantly.

In one embodiment, antibody fragments (i.e., for example, hMPV antibodyfragments), may include, but are not limited to, single chain antibodies(scFv or others) and can be produced/selected using phage displaytechnology. Although it is not necessary to understand the mechanism ofan invention, it is believed that the ability to express antibodyfragments on the surface of viruses that infect bacteria (bacteriophageor phage) makes it possible to isolate a single binding antibodyfragment from a library of greater than 10¹⁰ nonbinding clones. It isfurther believed that to express antibody fragments on the surface ofphage (phage display), an antibody fragment gene is inserted into thegene encoding a phage surface protein (pIII) and the antibodyfragment-pIII fusion protein is displayed on the phage surface.McCafferty et al., Nature 348: 552-554 (1990); and Hoogenboom et al.,Nucleic Acids Res. 19:4133-4137 (1991).

In one embodiment, functional antibody fragments on the surface of thephage can be used to separate out non-binding phage by antigen affinitychromatography. McCafferty et al., Nature 348:552-554 (1990). Althoughit is not necessary to understand the mechanism of an invention, it isbelieved that depending on the affinity of the antibody fragment,enrichment factors of 20 fold-1,000,000 fold may be obtained for asingle round of affinity selection. By infecting bacteria with theeluted phage, however, more phage can be grown and subjected to anotherround of selection. In this way, an enrichment of 1000 fold in one roundcan become 1,000,000 fold in two rounds of selection. Thus, even whenenrichments are low, multiple rounds of affinity selection can lead tothe isolation of rare phage. Marks et al., J. Mol. Biol. 222:581-597(1991). Since selection of the phage antibody library on antigen resultsin enrichment, the majority of clones bind antigen after as few as threeto four rounds of selection. Thus, only a relatively small number ofclones (several hundred) need to be analyzed for binding to antigen.

Naturally occurring human antibodies can be produced without priorimmunization by displaying very large and diverse V-gene repertoires onphage (Marks et al., supra). In one embodiment, natural V_(H) and V_(L)repertoires present in human peripheral blood lymphocytes are isolatedfrom unimmunized donors by PCR. The V-gene repertoires are splicedtogether at random using PCR to create a scFv gene repertoire which isthen cloned into a phage vector to create a library of 30 million phageantibodies. It is also possible to isolate antibodies against cellsurface antigens by selecting directly on intact cells. The antibodyfragments are highly specific for the antigen used for selection andhave affinities in the 1 μM to 100 nM range. Griffiths et al., EMBO J.12:725-734 (1993). Larger phage antibody libraries result in theisolation of more antibodies of higher binding affinity to a greaterproportion of antigens.

C. Proteins, Peptides and Fragments Thereof

In one embodiment, the binding partner can be a protein (i.e., forexample, a hMPV viral protein). In one embodiment, suitable proteins,peptides and fragments thereof include, but are not limited to, viralproteins (i.e., for example, M, L, N etc.), receptors (e.g., cellsurface receptors), receptor ligands (e.g., cytokines, growth factors,etc.), transcription factors and other nucleic acid binding proteins, aswell as members of binding pairs, such as biotin-avidin.

Binding proteins useful in the invention can be isolated from naturalsources, mutagenized from isolated proteins, or synthesized de novo.Isolation methods for naturally occurring proteins include, but are notlimited to, conventional protein purification methods including ammoniumsulfate precipitation, affinity chromatography, column chromatography,or gel electrophoresis. R. Scopes, (1982) In: Protein Purification,Springer-Verlag, N.Y.; and Deutscher (1990) Methods in Enzymology Vol.182: Guide to Protein Purification, Academic Press, Inc. N.Y. Althoughit is not necessary to understand the mechanism of an invention, it isbelieved that where a protein binds a target reversibly, affinitycolumns bearing the target can be used to affinity purify the protein.Alternatively the protein can be recombinantly expressed with a HIS-Tagand purified using Ni²⁺/NTA chromatography.

In another embodiment, a binding protein can be chemically synthesizedusing standard chemical peptide synthesis techniques. Where the desiredsubsequences are relatively short, the molecule may be synthesized as asingle contiguous polypeptide. Where larger molecules are desired,subsequences can be synthesized separately (in one or more units) andthen fused by condensation of the amino terminus of one molecule withthe carboxyl terminus of the other molecule thereby forming a peptidebond. This is typically accomplished using the same chemistry (e.g.,Fmoc, Tboc) used to couple single amino acids in commercial peptidesynthesizers.

In one embodiment, a solid phase synthesis may be used in which aC-terminal amino acid of a peptide sequence is attached to an insolublesupport. In one embodiment, the remaining amino acids in the sequenceare sequentially added. Barany et al., (1962) Solid-Phase PeptideSynthesis; pp. 3-284, In: The Peptides: Analysis, Synthesis, Biology.Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al.(1963) J. Am. Chem. Soc., 85: 2149-2156; and Stewart et al. (1984) SolidPhase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.

In one embodiment, a binding protein can also be produced usingrecombinant DNA methodology. Generally this involves generating a DNAsequence that encodes the binding protein, placing the DNA sequence inan expression cassette under the control of a particular promoter,expressing the protein in a host, isolating the expressed protein and,if necessary, renaturing the protein.

In one embodiment, DNA encoding for binding proteins can be prepared byany suitable method as described above, including, for example, cloningand restriction of appropriate sequences or direct chemical synthesis bymethods such as: i) the phosphotriester method of Narang et al. (1979)Meth. Enzymol. 68: 90-99; ii) the phosphodiester method of Brown et al.(1979) Meth. Enzymol. 68: 109 151; iii) the diethylphosphoramiditemethod of Beaucage et al. (1981) Tetra. Lett., 22: 1859 1862; and iv)the solid support method of U.S. Pat. No. 4,458,066 (all of which areherein incorporated by reference).

In one embodiment, DNA encoding a desired binding protein(s) can beexpressed in a variety of host cells, including, but not limited to, E.coli, other bacterial hosts, yeast, and various higher eukaryotic cells,such as the COS, CHO and HeLa cells lines and myeloma cell lines. TheDNA sequence encoding the binding protein is operably linked toappropriate expression control sequences for each host to produce anexpression construct. For E. coli, examples of appropriate expressioncontrol sequences include a promoter such as the T7, trp, or lambdapromoters, a ribosome binding site and preferably a transcriptiontermination signal. For eukaryotic cells, such control sequences caninclude a promoter, an enhancer derived, e.g., from immunoglobulingenes, SV40, cytomegalovirus, etc, and a polyadenylation sequence, andmay include splice donor and acceptor sequences.

In one embodiment, an expression vector can be transferred into thechosen host cell by such methods as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed with the expression vector can be selected byresistance to antibiotics conferred by genes contained on the plasmids,such as the amp, gpt, neo and hyg genes.

Once expressed, the recombinant binding proteins can be purified usingconventional techniques.

EXPERIMENTAL

The following examples are intended only as illustrative embodimentscontemplated by the present invention and should not be consideredlimiting in any respect.

Example I Preparation Of hMPV Hybridomas

This example provides one embodiment regarding the preparation ofhybridomas that produces monoclonal antibodies directed to hMPV. Thestains used below were based upon a sequencing and phylogenetic analysisof hMPV strains circulating in northern Italy in the 2001 through 2004winter-spring seasons. Gema et al, “Changing circulation rate of humanmetapneumovirus strains and styles among hospitalized pediatric patientsduring three consecutive winter-spring seasons” Arch. Virol. (In Press).

Viral strains belonging to hMPV types (A and B) and subtypes (A1-A2 andB1-B2) were isolated and propagated onto LLC-MK2 cell cultures. After 5to 10 passages, the various hMPV strains were released from infectedcultures. Infectivity was demonstrated by showing a 100% cytopathiceffect 5 days postinfection at titers of ≧10⁷ 50% tissue cultureinfective doses/ml. Following clarification, hMPV prototypes A and Bwere pelleted by ultracentrifugation, reaching titers of <10⁹ 50% tissueculture infective doses/ml.

These virus preparations were inoculated intramuscularly into BALB/cmice four times according to the following: i) the first virus inoculumwas administered using complete Freund's adjuvant; ii) three (3) weeksafter the first inoculum, the second inoculum was administered usingincomplete Freund's adjuvant; iii) five (5) weeks after the firstinoculum, the third inoculum was administered in saline; and iv) six (6)weeks after the first inoculum, the fourth inoculum was administered insaline.

A mouse spleen cell suspension was prepared and fused with Sp2/0Ag14myeloma cells. The hybridoma supernatants were tested for reactivitywith hMPV by enzyme-linked immunosorbent assay and the indirectfluorescent antibody (IFA) assay. Reactive hybridomas were identified,cloned and subcloned twice. Cross-reactivities with conventionalrespiratory viruses (i.e., for example, influenza viruses A and B,parainfluenza virus types 1 to 4, human respiratory syncytial virus,human adenoviruses, human coronaviruses 229E and OC43, and rhinoviruses)were tested by IFA assay. Hybridoma clones showing a variable degree ofcross-reactivity with human respiratory syncytial virus and wereexcluded from diagnostic use.

hMPV-specific MAbs showed three major IFA patterns on hMPV-infectedLLC-MK2 cell cultures: i) granular (FIGS. 1A and 1B; ii) filamentous(FIGS. 1C and 1D); and iii) foamy (FIGS. 1E and 1F). This pool of threeMAbs, including: i) clone C2C10 having reactivity with A1, A2, B1, andB2 hMPV subtype strains (immunoglobulin G1 [IgG1]), ii) clone C2D11having reactivity with A1, A2, B1, and B2 hMPV subtype strains (IgG1);and iii) clone T3H11 having reactivity with A1, A2, B1, and B2 hMPVsubtype strains (IgG2a), each representative of a different stainingpattern and reactive by both IFA and enzyme-linked immunosorbent assays,was used for diagnostic purposes. None of these hMPV MAbs were reactivewith avian metapneumoviruses A and B (data not shown; avianmetapneumovirus kindly provided by Ilaria Capua, IstitutoZooprofilattico delle Tre Venezie, Padua, Italy). Although it is notnecessary to understand the mechanism of an invention, it ishypothesized that the differences in staining patterns between thedifferent hMPV MAbs may be due to a selective affinity for differentviral proteins.

Example II Detection of hMPV Using Hybridoma Monoclonal Antibodies

Forty NPA samples collected during the winter-spring season of 2003-2004from 40 infants and young children admitted to the hospital because ofan episode of acute respiratory infection were retrospectively testedfor hMPV with MAbs by (i) using frozen smears of NPA samples for DFAstaining and (ii) inoculating frozen NPA samples, previously tested forconventional respiratory viruses, onto shell vial cell cultures.

Cells isolated from respiratory secretions had been previously used forpreparation of multiple smears, which were fixed with methanol-acetoneand stored at −80° C. After thawing, the smears were stained with MAbsto hMPV and, in parallel, with a high-titer guinea pig hyperimmuneserum. The pool of three MAbs was used for retrospective DFA staining ofthe 40 frozen NPA smears from respiratory secretions. See Table II; FIG.2A and FIG. 2B.

TABLE II Diagnostic value of DFA using MAbs to hMPV with respect toRT-PCR No. of samples tested Reactivity with by RT-PCR (%) MAbs to hMPVPositive Negative Total Positive 17 (73.9)  1 (5.9) 18 (45.0) Negative 6 (26.1) 16 (94.1) 22 (55.) Total 23^(a) 17^(b) 40^(c) ^(a)Positivepredictive value, 94.4%. ^(b)Negative predictive value, 72.7%.^(c)Agreement, 82.5%.

Specimens had been previously tested by RT-PCR for hMPV genes N and Fand found to be either positive (n=23; 18 type A and 5 type B strains)or negative (n=17). On the whole, DFA staining detected as positive17/23 NPAs found to be positive by RT-PCR (12 subtype A2, 2 subtype B1,and 3 subtype B2), while 6 subtype A2 strains were negative. Inaddition, DFA staining detected as negative 16/17 NPAs found to benegative by RT-PCR (one subtype A2 strain was positive by DFA stainingin very few cells). In the six RT-PCR-positive DFA-negative samples, nocorrelation was observed between a weak PCR signal and lack of DFAsignal, suggesting the presence of extracellular virus not detectable byDFA staining in the relevant NPAs.

In addition, four original NPAs (among those examined by DFA staining)stored frozen at −80° C. and previously found to be positive by RT-PCRwere inoculated onto LLC-MK2 shell vial cell cultures and incubated for48 h at 37° C. Following fixation with methanol-acetone andimmunostaining with the same pool of MAbs to hMPV, the four hMPV strainswere easily identified. Virus strains were detected in cell cultures aseither multiple single infected cells or small plaques with smallsyncytial formations. (FIG. 2C and FIG. 2D, respectively).

We claim:
 1. A composition comprising a solid support and one or morehuman metapneumovirus (hMPV)-specific monoclonal antibodies selectedfrom the group consisting of an hMPV monoclonal antibody produced byC2C10, an hMPV monoclonal antibody produced by F4A1, and an hMPVmonoclonal antibody produced by CB7F3.
 2. The composition of claim 1,wherein said solid support comprises a test strip.
 3. The composition ofclaim 2, wherein said test strip is attached to a dipstick.
 4. Thecomposition of claim 1, wherein said solid support comprises amicrotitre plate.
 5. The composition of claim 4, wherein said plate isfilled with said one or more hMPV-specific monoclonal antibodies.
 6. Thecomposition of claim 1, wherein said solid support comprises a cellbased assay.
 7. The composition of claim 6, wherein said cell basedassay comprises tMK cells.
 8. The composition of claim 1, wherein saidsolid support further comprises an elution electrophoresis system. 9.The composition of claim 8, wherein said elution electrophoresis systemcomprises a porous material under the influence of an electrical field.10. The composition of claim 9, wherein said porous material is selectedfrom at least one of the group consisting of dextran, cellulose,acrylamide, polymeric particles, and silica.
 11. The composition ofclaim 3, wherein said dipstick comprises at least one compound selectedfrom the group consisting of radioactive tracers, enzymes, colloidalmetals, and substrates.
 12. The composition of claim 1, wherein saidsolid support comprises a microfluidic device.
 13. The composition ofclaim 12, wherein said microfluidic device comprises a ferrofluid,wherein said antibodies are attached to said ferrofluid.
 14. Thecomposition of claim 12, wherein said microfluidic device comprises atleast one microchannel in fluidic communication with at least onemicrochamber.
 15. The composition of claim 2, wherein said test stripcomprises a fluid flow.
 16. The composition of claim 9, wherein saidporous material comprises a fluid flow.
 17. The composition of claim 12,wherein said microfluidic device comprises a fluid flow.
 18. Thecomposition of claim 2, wherein said test strip comprisesnitrocellulose.
 19. The composition of claim 1, wherein said one or morehuman metapneumovirus (hMPV)-specific monoclonal antibodies arefluorescently labeled.
 20. The composition of claim 1, wherein saidsolid support further comprises a secondary anti-mouse antibody.
 21. Thecomposition of claim 20, wherein said secondary anti-mouse antibody iscoupled with an enzyme/substrate complex.
 22. The composition of claim21, wherein said enzyme/substrate complex is a peroxidase-labeledanti-mouse IgG antibody and a peroxidase substrate.
 23. A method fordetecting the presence of human metapneumovirus in a sample, comprising:contacting said sample with a solid support and one or more humanmetapneumovirus (hMPV)-specific monoclonal antibodies selected from thegroup consisting of an hMPV monoclonal antibody produced by C2C10, anhMPV monoclonal antibody produced by F4A1, and an hMPV monoclonalantibody produced by CB7F3, wherein said sample is contacted with saidsolid support under conditions such that said one or more hMPV-specificmonoclonal antibodies binds to said human metapneumovirus.
 24. Themethod of claim 23, wherein said solid support comprises a test strip.25. The method of claim 24, wherein said test strip is attached to adipstick.
 26. The method of claim 23, wherein said solid supportcomprises a microtitre plate.
 27. The method of claim 26, wherein saidmicrotitre plate is filled with said one or more hMPV-specificmonoclonal antibodies.
 28. The method of claim 23, wherein said assaycomprises a cell based assay.
 29. The method of claim 28, wherein saidcell based assay comprises tMK cells.
 30. The method of claim 23,wherein said solid support further comprises an elution electrophoresissystem.
 31. The method of claim 30, wherein said elution electrophoresissystem comprises a porous material under the influence of an electricalfield.
 32. The method of claim 31, wherein said porous material isselected from at least one of the group consisting of dextran,cellulose, acrylamide, polymeric particles, and silica.
 33. The methodof claim 25, wherein said dipstick comprises at least one compoundselected from the group consisting of radioactive tracers, enzymes,colloidal metals, and substrates.
 34. The method of claim 23, whereinsaid solid support comprises a microfluidic device.
 35. The method ofclaim 34, wherein said microfluidic device comprises a ferrofluid,wherein said antibodies are attached to said ferrofluid.
 36. The methodof claim 34, wherein said microfluidic device comprises at least onemicrochannel in fluidic communication with at least one microchamber.37. The method of claim 24, wherein said test strip comprises a fluidflow.
 38. The method of claim 31, wherein said porous material comprisesa fluid flow.
 39. The method of claim 34, wherein said microfluidicdevice comprises a fluid flow.
 40. The method of claim 24, wherein saidtest strip comprises nitrocellulose.
 41. The method of claim 23, whereinsaid one or more human metapneumovirus (hMPV)-specific monoclonalantibodies are fluorescently labeled.
 42. The method of claim 23,wherein said solid support further comprises a secondary anti-mouseantibody.
 43. The method of claim 42, wherein said secondary anti-mouseantibody is coupled with an enzyme/substrate complex.
 44. The method ofclaim 43, wherein said enzyme/substrate complex is a peroxidase-labeledanti-mouse IgG antibody and a peroxidase substrate.